Human mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into many of the cells resident in musculoskeletal and stromal tissues of the human body, including fibroblasts, chondrocytes, osteoblasts, myocytes, and adipocytes. While differentiation of the MSCs into appropriate lineages may enhance healing of injured tissues, inappropriate lineage specification may be responsible for numerous pathophysiologic processes, including the decreased bone and increased fat in osteoporotic bones, and the calcification of atherosclerotic vessel walls. Regulation of the lineage commitment of MSCs by local microenvironmental cues therefore may be critical to our fundamental understanding of numerous degenerative as well as healing processes. The long term objective of this research is to characterize the cues within the local surrounding microenvironment that drive the lineage specification and differentiation of human mesenchymal stem cells (MSCs), and the molecular pathways involved. The investigator has discovered that adhesion of MSCs to fibronectin regulates a commitment switch in the MSCs between adipogenic and osteogenic lineage specification, through a mechanism involving RhoA signaling and cytoskeletal tension. During the past grant period, he has demonstrated that these adhesive and mechanical cues specifically regulate SMAD and PPARgamma, two key transcription factors critical to osteogenesis and adipogenesis. Specific Aim 1 will be to investigate the how integrin activation regulates BMP-SMAD signaling and osteogenesis. Specific Aim 2 will be to investigate how RhoA regulates SMAD activity. Specific Aim 3 will be to investigate how adhesive and mechanical cues regulate PPARgamma signaling. Together, these studies will define roles of cell adhesion, RhoA, and cytoskeletal tension in MSC lineage commitment, and establish a molecular basis for the regulation of MSC differentiation by microenvironmental cues. PUBLIC HEALTH RELEVANCE: Human mesenchymal stem cells contribute to the maintenance and healing of many musculoskeletal tissues, but they also can produce inappropriate cell lineages to exacerbate disease, such as occurs with calcification of atherosclerotic vessels. They are now being isolated as a promising source of stem cells for regenerative therapies, but again their utility rests upon predictable control of their differentiation potential. This project is designed to develop a better understanding of how adhesive and mechanical cues direct these stem cells to differentiate into specific lineages, such that we may better design future approaches to treat or prevent degenerative diseases.